Y. Tanaka performed the technical improvement of Y2H. This work is supported by JSPS KAKENHI Grant Number JP22K06336 and the Institute for Fermentation, Osaka.

1. Construction of the mutant library
<ol>
	<li>Set up the polymerase chain reaction (PCR) (an example is shown below). Generally, prepare 50 &#181;L of the reaction, which is divided into ten aliquots of 5 &#181;L, and amplify the target gene fragment in a PCR machine4. 
	NOTE: Users should select an appropriate polymerase to efficiently introduce mutations. If the DNA fragment to be amplified is short, then a polymerase with a higher error rate, such as Taq polymerase, which lacks proofreading activity, is needed. If the DNA fragment to be amplified is long, then a higher fidelity polymerase is needed to reduce the number of mutations. Mutations occur every few hundred base pairs after 30 cycles of amplification with regular Taq polymerase4. In the Representative results, a different Taq polymerase was used (see Table of Materials), which has higher fidelity than regular Taq polymerase, and the mutation rate was one in 600 base pairs. An example of the PCR mixtures, the primer details, and the reaction conditions are provided in Supplementary File 1.</li>
	<li>When the PCR is completed, combine all the aliquots of the reaction, confirm that the DNA fragments of interest have been amplified using agarose gel electrophoresis, etc., and purify the DNA5.</li>
	<li>Assemble the DNA fragment generated in step 1.2 with the appropriate Y2H vector using a &#39;seamless&#39; cloning strategy (Figure 1). 
	NOTE: The seamless cloning system, such as Gibson assembly6, minimizes contamination of the constructed mutant library by empty vectors generated via self-ligation. A list of Y2H vectors is provided in Table 1. They can be prepared by BamHI digestion before assembly. All plasmids are available from the National BioResource Project - yeast (see Table of Materials).</li>
	<li>Introduce the reaction mixture generated in step 1.3 into Escherichia coli competent cells prepared according to a highly efficient E. coli transformation protocol (e.g., Inoue et al.7). Spread all competent cells on several LB plates (1% tryptone, 0.5% yeast extract, 1% NaCl, and 2% agar) containing appropriate antibiotics (see Table of Materials). At this point, spread a small amount (~1/100 of the total reaction) onto the same selection plate to calculate the library titer. Incubate the plates at 37 &#176;C overnight.</li>
	<li>Once a large number of E. coli colonies have appeared, scrape all cells from the surface of the plate using a disposable plastic loop. Recover plasmid DNA from these cells using popular methods, such as alkaline lysis8. At the same time, count the number of colonies that appear after spreading small aliquots of the transformation mixture on the plate. Using this number, estimate the total number of independent clones in the library. 
	NOTE: At this point, pick up a few independent colonies (4-6) that appear on the plate on which the small aliquots of transformation reaction were spread, culture them separately, and isolate plasmids from them to confirm that virtually all clones carry the desired DNA fragments5. Many commercial kits are available to isolate plasmids from E. coli. The number of colonies that appear on the plate after spreading small aliquots can be counted manually.</li>
	<li>Measure the concentration of the library DNA pool. Usually, a few hundred nanograms of library DNA is sufficient to obtain several thousand transformants in the next step. 
	NOTE: It is recommended to measure the DNA concentration using a fluorescent dye that binds to DNA because measurement of A260 is frequently inaccurate.</li>
</ol>
2. Transformation of yeast with the mutant library and replica plating
<ol>
	<li>Introduce the mutant library into the host yeast strain (TAT-7 (L40-ura3)9,10 MATa, leu2-3,112, trp1-901, his3-&#916;200, ade2-101, gal80&#916;, LYS2:(lexAop)4-HIS3, ura3:(lexAop)8-lacZ) for the Y2H assay using about 100 ng of DNA. Pre-transform the host cells with the &#39;bait&#39; plasmid.
	<ol>
		<li>After the transformation procedure, suspend yeast cells in sterile water and spread aliquots of different amounts on appropriate plates (usually SC medium lacking leucine and tryptophan: SC-LW [0.67% yeast nitrogen base, 2% glucose, 1.546 g/L SC double drop-out mix -Leu -Trp, and 2% agar, see Table of Materials]). Incubate these plates overnight at 30 &#176;C. 
		NOTE: The standard protocol, such as the lithium acetate-PEG method11 with ~5 &#215; 106 logarithmically growing cells, is sufficient for the transformation of yeast. Another method with a high transformation efficiency, such as electroporation, can also be used.</li>
	</ol>
	</li>
	<li>The next day, when tiny colonies appear, select the plates with an appropriate number of cells (500-2000) and make replicates from them as follows.</li>
	<li>Set two sheets of filter paper on a replica block to make several replicas (medium is SC-LW and SC-LW containing kanamycin) (see Table of Materials). Incubate at 30 &#176;C for 1-2 days. 
	NOTE: The concentration of kanamycin (G418) is 600 &#181;g/mL. Do not use velvet for replica plating because the resolution of colonies will be lost.</li>
	<li>Once colonies have grown, compare plates and pick candidates. Perform a Y2H color assay (see step 3) if lacZ is used as the reporter. 
	NOTE: For the Y2H reporter, lacZ is usually better at detecting a weak interaction than HIS3.</li>
</ol>
3. Y2H color assay
<ol>
	<li>Place a sheet of filter paper cut in advance to the appropriate size on the replica plate prepared using step 2. Be careful not to allow air bubbles to form between the filter paper and the plate. 
	NOTE: Use No. 4A filter paper or Grade 50 filter paper (see Table of Materials). Any SC-LW or SC-LW containing kanamycin plates, which are made by replica plating, can be used for the color assay.</li>
	<li>When the filter paper on the plate is completely moistened (when the color of the filter paper changes completely), place several sheets of paper towel on top and make it come into contact with the filter paper to remove excess moisture. Remove the paper towel when it absorbs the water and changes color. Repeat this process two or three times.</li>
	<li>Pick up the edges of the filter paper with tweezers, peel it off the plate quickly, immerse it in liquid nitrogen, and freeze it. If liquid nitrogen is not available, place the filter paper on a plastic tray with the colony side up and immediately place it in a freezer (-20 &#176;C to -80 &#176;C) to freeze completely.</li>
	<li>Remove the frozen filter paper and place it, with the colony side up, on a dry paper towel to thaw. Immediately after thawing, place the filter paper, with the colony side up, on another filter paper that has been placed on a plastic tray or sealable container and soaked with Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, and 1 mM MgSO4, pH 7.0) containing X-gal (5-bromo-5-chloro-3-indolyl-&#946;-D-galactoside) and 2-mercaptoethanol9 (see Table of Materials). Be careful not to allow air bubbles to form between the top and bottom filter papers.</li>
	<li>Cover the plastic tray containing the filter paper and put it into an airtight container or plastic bag. Close the airtight container or plastic bag and incubate it at 37 &#176;C until a blue color appears.</li>
	<li>When a blue color appears, place the filter paper with the colony side up on about 500 &#181;L of stop solution (1 M Na2CO3) placed on a clean plastic tray. Stop solution soaks in quickly; therefore, remove the filter immediately, place it on a fresh paper towel with the colony side up, and allow it to dry.</li>
	<li>After the paper towel is replaced and the filter is dry enough, use a scanner or other device to capture the data.</li>
</ol>
4. Recovery and confirmation of candidate clones
<ol>
	<li>Select white and KanR candidate clones from the replica&#39;s original plate (or the replicated plate not used for the color assay). Examples are shown in Figure 1C. Confirm that candidate clones are white and KanR by re-streaking them as a small patch on SC-LW medium containing kanamycin and incubating them at 30 &#176;C for 1-2 days. Make at least two sets or make replicates the next day. 
	NOTE: White colonies should be selected because pale blue colonies might retain the interaction.</li>
	<li>Once candidate clones have re-grown well, repeat the color assay with at least one plate generated in step 4.1 as described in step 3 and confirm that they remain white and do not turn blue (Figure 2A). 
	NOTE: When re-streaking the candidates, do not carry over large amounts of cells. Too many cells make it impossible to distinguish KanR and KanS clones.</li>
	<li>Take clones that are still white and KanR generated in step 4.2 from the plate remains and grow them independently in 1 mL of selection medium (SC-L or SC-W, depending on which vector is used for the library construction) overnight at 30 &#176;C.</li>
	<li>Transfer the culture to a microtube and collect cells by centrifugation (~15,000 &#215; g, for 10-30 seconds&#160;at room temperature). Isolate whole DNA from the cell pellet as follows.</li>
	<li>Suspend the cell pellet in 400 &#181;L of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl (8.0), and 1 mM EDTA) containing 1 &#181;L each of 2-mercaptoethanol and 20 mg/mL Zymolyase 100T (see Table of Materials), and incubate at 37 &#176;C for 30 min to digest cell walls. At this time, mix gently by inverting the tube every 5-10 min.</li>
	<li>When the cell suspension has become opaque or translucent, add an equal volume of a mixture of phenol-chloroform-isoamyl alcohol (25:24:1) and mix well by vortexing at moderate speed for 10-20 s.</li>
	<li>Centrifuge the microtubes in a microcentrifuge for 5 min at full speed (~15,000 &#215; g,&#160;at room temperature) and recover the aqueous phase containing DNA in a new microtube. Add 0.8 volume of isopropanol and mix well by inverting the tube.</li>
	<li>Leave the tube at room temperature for 2-3 min and then centrifuge in a microcentrifuge for 4-5 min at full speed (~15,000 &#215; g, at room temperature) to precipitate the DNA.</li>
	<li>Remove the supernatant and wash the precipitate with about 500 &#181;L of 70% ethanol. Remove ethanol completely and dry the precipitate briefly.</li>
	<li>Add 20 &#181;L of TE buffer (10 mM Tris-Cl (8.0) and 1 mM EDTA) to the precipitate, mix briefly, and leave the tube overnight at room temperature to dissolve the precipitate. 
	NOTE: If users are in a hurry, keep tubes at 37-50 &#176;C. The DNA precipitate will dissolve in a few hours.</li>
	<li>Once the pellet is completely dissolved, transform E. coli with a small amount of this DNA solution. 
	NOTE: About 0.5 &#181;L of DNA solution is sufficient if E. coli with a high transformation efficiency is used.</li>
	<li>When E. coli colonies appear, pick several independent colonies, culture them, and recover plasmids by standard methods, such as alkaline lysis.</li>
	<li>Introduce the plasmid DNA obtained in step 4.12 into Y2H host cells harboring the bait plasmid. When transformants appear, check them by the color assay (they should appear white) and western blotting4 (they should express a protein of the desired size). 
	NOTE: The post-alkaline method12 is an easy and rapid way to prepare a whole-cell extract from yeast.</li>
	<li>If the above two criteria are met, determine the mutation site of the clones by nucleotide sequencing4.</li>
</ol>

Biomolecular Interaction Studies Using Yeast Two-Hybrid Technique 

<ol>
	<li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Leanna, C. A., Hannink, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reverse+two-hybrid+system:+A+genetic+scheme+for+selection+against+specific+protein/protein+interactions.">The reverse two-hybrid system: A genetic scheme for selection against specific protein/protein interactions.</a> Nucleic Acids Res. 24 (17), 3341-3347 (1996).</li><li>Vidal, M., Brachmann, R. K., Fattaey, A., Harlow, E., Boeke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+two-hybrid+and+one-hybrid+systems+to+detect+dissociation+of+protein-protein+and+DNA-protein+interactions.">Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.</a> Proc Natl Acad Sci U S A. 93 (19), 10315-10320 (1996).</li><li>Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+the+isolation+of+interaction-null/impaired+mutants+using+the+yeast+two-hybrid+technique.">An efficient method for the isolation of interaction-null/impaired mutants using the yeast two-hybrid technique.</a> Genes Cells. 24 (12), 781-788 (2019).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: A laboratory manual. 4th edn. , (2012).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96 (1), 23-28 (1990).</li><li>Birnboim, H. C., Doly, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+alkaline+extraction+procedure+for+screening+recombinant+plasmid+DNA.">A rapid alkaline extraction procedure for screening recombinant plasmid DNA.</a> Nucleic Acids Res. 7 (6), 1513-1523 (1979).</li><li>Bartel, P. L., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein-protein+interactions+using+two-hybrid+system.">Analyzing protein-protein interactions using two-hybrid system.</a> Meth Enzymol. 254, 241-263 (1995).</li><li>Hollenberg, S. M., Sternglanz, R., Cheng, P. F., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+family+of+tissue-specific+basic+helix-loop-helix+proteins+with+a+two-hybrid+system.">Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.</a> Mol Cell Biol. 15 (7), 3813-3822 (1995).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+yeast+by+lithium+acetate/single-stranded+carrier+DNA/polyethylene+glycol+method.">Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.</a> Meth Enzymol. 350, 87-96 (2002).</li><li>Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+reliable+protein+extraction+from+yeast.">Rapid and reliable protein extraction from yeast.</a> Yeast. 16 (9), 857-860 (2000).</li><li>Van Deursen, F., Sengupta, S., De Piccoli, G., Sanchez-Diaz, A., Labib, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mcm10+associates+with+the+loaded+DNA+helicase+at+replication+origins+and+defines+a+novel+step+in+its+activation.">Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation.</a> EMBO J. 31 (9), 2195-2206 (2012).</li><li>Watase, G., Takisawa, H., Kanemaki, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mcm10+plays+a+role+in+functioning+of+the+eukaryotic+replicative+DNA+helicase,+cdc45-mcm-gins.">Mcm10 plays a role in functioning of the eukaryotic replicative DNA helicase, cdc45-mcm-gins.</a> Current Biology: CB. 22 (4), 343-349 (2012).</li><li>Miyazawa-Onami, M., Araki, H., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-initiation+complex+assembly+functions+as+a+molecular+switch+that+splits+the+mcm2-7+double+hexamer.">Pre-initiation complex assembly functions as a molecular switch that splits the mcm2-7 double hexamer.</a> EMBO Rep. 18 (10), 1752-1761 (2017).</li></ol>

The authors declare that they have no conflict of interest.

This article describes how to isolate interaction-null/impaired mutants using the Y2H assay. Such mutants are powerful tools to analyze the function of a protein of interest. To isolate such mutants, rY2H assays were developed previously by modifying the Y2H host strain2,3. However, they have not greatly reduced the amount of labor. By contrast, mutants can be isolated with this method without a significant amount of labor. In this method, modification of the Y2H...

Interactions between biomolecules are the most basic part of biological phenomena. Protein-protein interactions constitute a significant part of such interactions. Therefore, identification of the interaction partner(s) of a protein of interest is critical to further elucidate the function of the protein/gene of interest. The yeast two-hybrid (Y2H) method is a popular technique to identify protein-protein interactions in vivo1. In this system, two proteins (X and Y) whose interaction is to be tested are fused to the DNA-binding (DB) domain and transcriptional activation domain (AD), respectively. The DB-X fusion protein binds to a recognition sequence of the DB domain; therefore, when proteins X and Y interact, the AD-Y fusion protein comes into the proximity of the recognition sequence. Consequently, transcription of the reporter gene downstream of the recognition sequence is activated. Therefore, the presence or absence of reporter gene activity can be used to determine the presence or absence of the protein-protein interaction1.
Once a specific interaction partner of the protein of interest is identified, further analyses should be performed to elucidate the biological function of the interaction. For this purpose, if mutants of the proteins that impair or remove the specific protein-protein interaction can be isolated, they will serve as powerful tools. The Y2H system can be used directly to isolate such mutants by screening 'interaction-negative' clones, starting with the wild-type 'interaction-positive' clone. To accelerate this process, 'reverse' Y2H (rY2H) systems were developed2,3. In rY2H systems, the host yeast strains harbor counter-selectable marker genes as reporter genes, meaning yeast cells grow only when the AD-Y and DB-X proteins do not interact.
Although both the Y2H and rY2H systems allow the isolation of interaction-negative mutants, the process of isolating the mutants is laborious because not all of the candidates obtained by screening carry the desired type of mutations (usually missense mutations). The most serious issue is that a significant fraction of candidates harbor frameshift or nonsense mutations, and it is necessary to perform western blotting to exclude undesired clones. To overcome this problem, new plasmid vectors have been developed4. In these vectors, KanMX, a drug resistance marker, is positioned out-of-frame downstream of the DB domain or AD. The marker gene becomes in-frame with the DB domain or AD only when the gene of interest is inserted. When a random mutation(s) is introduced in the gene of interest, undesirable mutants, such as those with frameshift or nonsense mutations, can be easily eliminated by performing drug resistance selection, and candidates carrying desirable missense mutations can be easily identified with the Y2H screen4. This article presents a protocol to isolate interaction-null/impaired mutants of a protein of interest using this strategy.

<table><tbody><tr><td>0.5 M EDTA (8.0)</td><td>Nacalai Tesque Inc.</td><td>14347-21</td><td></td></tr><tr><td>10% SDS Solution</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>313-90275</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>135-07522</td><td></td></tr><tr><td>2-propanol</td><td>Kishida Chemical Co. Ltd.</td><td>110-64785</td><td></td></tr><tr><td>5-Bromo-4-chloro-3-indolyl-&amp;beta;-D-galactopyranoside (X-Gal)</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>021-07852</td><td></td></tr><tr><td>Agar</td><td>Formedium</td><td>AGR60</td><td></td></tr><tr><td>Ampicillin Sodium</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>68-52-3</td><td></td></tr><tr><td>Anti-HA-tag mAb-HRP-DirecT&amp;nbsp;&amp;nbsp;</td><td>Medical &amp;amp; Biological Laboratories Co. Ltd.</td><td>M180-7</td><td></td></tr><tr><td>DNA from salmon testes</td><td>Merck KGaA.</td><td>D1626</td><td></td></tr><tr><td>Ethanol</td><td>Merck KGaA.</td><td>9-0770-4-4L-J</td><td></td></tr><tr><td>Filter paper for colony lift (Grade 50)</td><td>Whatman, Cytiva</td><td>1450-090</td><td></td></tr><tr><td>Filter paper for colony lift (No.4A)</td><td>Advantec Toyo Kaisha, Ltd.</td><td>01411090</td><td></td></tr><tr><td>Filter paper for replicaplating (No.1)</td><td>Advantec Toyo Kaisha, Ltd.</td><td>00011150</td><td></td></tr><tr><td>G-418 Sulfate</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>075-05962</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Kishida Chemical Co. Ltd.</td><td>230-37585</td><td></td></tr><tr><td>KCl</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>163-03545</td><td></td></tr><tr><td>Lithium Acetate Dihydrate</td><td>Nacalai Tesque Inc.</td><td>20604-22</td><td></td></tr><tr><td>MgSO4&amp;bull;7H2O</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>131-00405</td><td></td></tr><tr><td>Na2HPO4&amp;bull;12H2O</td><td>Nacalai Tesque Inc.</td><td>10039-32-4</td><td></td></tr><tr><td>NaCl</td><td>Nacalai Tesque Inc.</td><td>31319-45</td><td></td></tr><tr><td>NaH2PO4&amp;bull;2H2O</td><td>Nacalai Tesque Inc.</td><td>31717-25</td><td></td></tr><tr><td>Paper towel</td><td>AS ONE Corp.</td><td>7-6200-02</td><td></td></tr><tr><td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1</td><td>Nacalai Tesque Inc.</td><td>25970-56</td><td></td></tr><tr><td>Plasmid DNAs</td><td>the National BioResource Project - yeast (https://yeast.nig.ac.jp/yeast/top.xhtml)</td><td></td><td></td></tr><tr><td>Plasmid isolation Kit</td><td>Nippon Genetics Co. Ltd.</td><td>FG-90502</td><td></td></tr><tr><td>Polyethylene Glycol #4,000</td><td>Nacalai Tesque Inc.</td><td>11574-15</td><td></td></tr><tr><td>SC double drop-out mix -Leu -Trp</td><td>Formedium</td><td>DSCK172</td><td></td></tr><tr><td>Seamless cloning kit (In-Fusion assembly )</td><td>Takara Bio Inc.</td><td>#639648</td><td></td></tr><tr><td>Skim milk powder</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>190-12865</td><td></td></tr><tr><td>Streptomycin Sulfate</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>3810-74-0</td><td></td></tr><tr><td>Taq polymerase (GoTaq Green Master Mixes)</td><td>Promega Corp.</td><td>M7122</td><td></td></tr><tr><td>TRIS (hydroxymethyl) aminomethane</td><td>Formedium</td><td>TRIS01</td><td></td></tr><tr><td>Triton X-100</td><td>Nacalai Tesque Inc.</td><td>12967-45</td><td></td></tr><tr><td>Tryptone</td><td>ThermoFisher scientific Inc.</td><td>211705</td><td></td></tr><tr><td>Tween 20</td><td>Nacalai Tesque Inc.</td><td>35624-15</td><td></td></tr><tr><td>Yeast Extract</td><td>ThermoFisher scientific Inc.</td><td>212750</td><td></td></tr><tr><td>Yeast Nitrogen Base (YNB)</td><td>Formedium</td><td>CYN0210</td><td></td></tr><tr><td>Zymolyase 100T</td><td>Nacalai Tesque Inc.</td><td>07665-55</td><td></td></tr></tbody></table>

isolating interaction-null&#47;impaired mutants using the yeast two-hybrid assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

Recently, it was found that the C-terminal half of Pol2 protein (Pol2-C) interacts with Mcm10. Both proteins are essential for the initiation of DNA replication and, hence, for cell growth in the budding yeast Saccharomyces cerevisiae13,14,15. To help understand the biological significance of this interaction, Pol2-C mutants that have no/diminished interaction with Mcm10 were isolated using the method described here.</p...

Watch this Scientific Journal Video about Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast at JoVE.com

Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...

isolating-interaction-nullimpaired-mutants-using-yeast-two-hybrid

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347 Views.  Kochi University of Technology. The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Video: Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Biochemistry

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Genetics

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Immunology and Infection

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Modified Yeast-One Hybrid Assay to Detect Heteromeric Protein Complex-DNA Interactions

Source: Tripathi, P. et al., <a href="https://app.jove.com/v/56080">A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the modified yeast one-hybrid assay, which detects multiple proteins interacting with target DNA. The transformed yeast cells contain the target DNA sequence upstream of the reporter gene. Additionally, they express transcription factor proteins. When the proteins form complexes and bind to the target DNA, the reporter gene is expressed and can be detected enzymatically.

Source: Tripathi, P. et al., A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies. J. Vis. Exp. (2017)
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Isolating Interaction-Null/Impaired Mutants Using the Yeast Two-Hybrid Assay

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Chapters in this video

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Modified Yeast-One Hybrid Assay to Detect Heteromeric Protein Complex-DNA Interactions